Haruo Kanatani

Maturation-inducing substance in starfishes

Publisher Summary This chapter discusses the maturation-inducing substance in starfishes. It reviews the mechanism of reproductive phenomena in starfishes based on the physiological activities of two substances, a gonad-stimulating hormonal peptide of neural origin and a maturation-inducing substance produced in the gonad under the influence of the former. The mechanism of gamete release and oocyte maturation in starfishes, based on the action of gonad-stimulating substance (GSS) and maturation-inducing substance (MIS), I-methyladenine, is summarized in the chapter. These reproductive phenomena are effected under hormonal influence in starfishes. Once GSS is liberated from the nerves, it reaches the celomic cavity and enters the ovary, and the cells of the follicles around the oocytes respond to GSS by producing I-methyladenine. The starfish gonad contains a substance that suppresses cell division of Chaetopterus and Arbacia eggs and seems to block oocyte meiosis in starfishes. Starfish oocytes remain in the germinal vesicle stage until just before spawning occurs, whereas spermatogenesis proceeds all the way to the formation of spermatozoa long before the release of sperm. However, the localization in the ovary, chemical nature, and mechanism of action under physiological conditions of such a substance are still obscure.

ROLE OF FOLLICLE CELLS IN MATURATION OF STARFISH OOCYTES

The role of the follicles in oocyte maturation was studied with the starfish, Asterina pectinifera. Follicles, obtained from isolated oocytes with follicular envelopes, produced maturation-inducing substance (MIS) when incubated in sea water containing gonad-stimulating substance (GSS). Isolated oocytes without follicular envelopes were found to have no such capacity to produce MIS even in the presence of GSS. MIS produced by follicles under the influence of GSS was extracted with 95% ethanol and purified on a Sephadex G-15 column. This MIS was identified as 1-methyladenine with thin layer chromatography, showing that the follicles can produce 1-methyladenine under the influence of GSS. MIS was also produced by follicles when they were incubated in sea water containing 1-methyladenosine in the absence of GSS. This indicates that I-methyladenosine ribohydrolase, which splits l-methyladenosine to 1-methyladenine and ribose, is present in these follicles. The capacity of follicles to produce MIS under the influence of GSS was found to depend on the age of oocytes from which the follicles were isolated; follicles taken from young oocytes failed to produce MIS even in the presence of GSS. 1-methyladenosine ribohydrolase was found to be present even in young follicles, although its activity was lower than that of the follicles of full-grown oocytes.

Spawning of Starfish: Action of Gamete-Shedding Substance Obtained from Radial Nerves.

An extract of the radial nerves of the starfish, Asterias amurensis, acts on the ovary in two ways: it induces meiosis and brings on spawning. Contraction of the gonadal wall, the, driving force for spawning, does not happen until this gamete-shedding substance acts to liberate the eggs that adhere to each other or to the inner surface of the gonadal wall.

MECHANISM OF STARFISH SPAWNING. III. PROPERTIES AND ACTION OF MEIOSIS‐INDUCING SUBSTANCE PRODUCED IN GONAD UNDER INFLUENCE OF GONAD‐STIMULATING SUBSTANCE

The gonad‐stimulating substance (GSS) contained in nerve extract of the starfish, Asterina pectinifera, acts on the ovary to produce an active substance responsible for oocyte maturation, meiosis‐inducing substance (MIS). MIS was successfully separated from GSS by gel‐filtration on Sephadex. The MIS fraction had also spawning‐inducing activity. Starfish testis also produced MIS under the influence of GSS. MIS production was shown in six starfish species. Although some species specificity characterizes GSS, this was not observed in MIS.

Site of production of meiosis-inducing substance in ovary of starfish☆

The site of production of meiosis-inducing substance (MIS), produced in the ovary under the influence of gonad-stimulating substance (GSS) taken from radial nerves, was studied with the starfish, Asterina pectinifera. The rate of oocyte maturation observed in isolated oocytes with follicles kept in sea water containing GSS (100 μg/ml) was generally low when a small number of eggs per unit quantity of sea water was used. However, with increased number of oocytes per milliliter of GSS-sea water, the maturation rate increased up to 100% (104 eggs/ml). The supernatant of such an incubation mixture of oocytes and GSS contained an appreciable amount of MIS. When a large number of oocytes (104/ml) was used, the rate of oocyte maturation increased with the rise in concentration of GSS. Isolated follicles were found to produce MIS when incubated in sea water containing GSS, suggesting that the site of production of MIS is the follicle cells. The physiological role of the follicle cells surrounding the full grown oocytes seems to be to produce MIS just before spawning under the influence of GSS.

MECHANISM OF STARFISH SPAWNING. II. SOME ASPECTS OF ACTION OF A NEURAL SUBSTANCE OBTAINED FROM RADIAL NERVE

1. When a water extract of radial nerves of the starfish, Asterias amurensis, was locally applied to one part of an isolated whole ovary for an appropriate period and then small slits were made on the ovarian wall, intense discharge of eggs occurred only in the treated portion.2. Eggs within the nontreated part of the ovary were observed to adhere to each other and to the gonadal wall by means of the follicle layer surrounding them.3. Eggs within ligated ovarian fragments which had been treated with nerve extract lost their follicles and underwent maturation. These eggs were found to be freely movable.4. Artificial spawning could be induced without using nerve extract; ovarian fragments immersed in Mg-free sea water released their eggs after a certain interval, while those treated with Ca-free sea water for an appropriate period spawn after subsequent addition of calcium.5. Breakdown of follicles occurred within an ovary treated with calcium-free sea water.6. Treatment with contraction-inducing agents suc...

Induction of cytoplasmic maturation by 1-methyladenine in starfish oocytes after removal of the germinal vesicle

Abstract 1-Methyladenine induces cytoplasmic maturation as revealed by the elevation of fertilization membrane both in oocytes from which germinal vesicles were removed and in fragments of the enucleated oocytes. However, at the most only abortive cleavage occurs in the enucleated oocytes. Thus, fertilizability of oocytes can be achieved with 1-methyladenine treatment but without any participation of germinal vesicle material, though it appears that mixing of germinal vesicle material with egg cytoplasm is a necessary prerequisite for genuine cleavage.

Induction of starfish oocyte maturation by disulfide-reducing agents

Abstract Oocyte maturation was found to be induced by disulfide-reducing agents such as dithiothreitol (DTT) and 2,3-dimercapto-1-propanol (BAL) in the starfish, Asterina pectinifera . The follicular envelopes around the oocytes broke and retracted into small clumps of cells on treatment with these reagents, as in the case of 1-methyladenine. Upon insemination, fertilizable eggs obtained by treatment with DTT formed a tight fertilization membrane and underwent cleavage. Such eggs developed normally to bipinnaria larvae. Cysteine and glutathione-SH had no effect in inducing oocyte maturation. On the other hand, pretreatment with sulfhydryl reagents such as p -chloromercurybenzoate (PCMB), iodoacetamide (IAM) and N -ethylmaleimide (NEM) completely suppressed 1-methyladenine-induced oocyte maturation. This inhibitory effect of sulfhydryl reagents on oocyte maturation was diminished by subsequent treatment with DTT or BAL with or without 1-methyladenine. Pretreatment with o -iodosobenzoate failed to inhibit 1-methyladenine-induced oocyte maturation.

PURIFICATION OF GONAD‐STIMULATING SUBSTANCE OBTAINED FROM RADIAL NERVES OF THE STARFISH, ASTERIAS AMURENSIS

Purification of starfish gonad‐stimulating substance (GSS), which induces shedding of gametes and oocyte maturation, was carried out using lyophilized radial nerves of Asterias amurensis as source material. In the first series of experiments, 1.3 mg of the purified GSS, which induced spawning at a concentration of 0.0096 μg/ml, was isolated from acetone powder of lyophilized radial nerves of 7,360 starfish through several steps of purification procedures consisting of gel‐filtrations on Sephadex G–50 and G–25 columns of various sizes and ion‐exchange chromatography on DEAE‐Sephadex columns by gradient as well as step‐wise elution. With this sample, the molecular weight and amino acid composition of GSS were estimated. Another series of experiments, conducted on a similar amount of material with purification procedures which were essentially the same as those of the first series except for employing 2 steps of partition chromatography instead of extensive gel‐filtration, gave about 0.1 mg of purified sample which served as material for studies of the amino acid composition and electrophoretic properties of GSS.

Starfish oocyte maturation and reduction of disulfide-bond on oocyte surface.

Abstract Disulfide-reducing agents such as DTT and 2,3-dimercapto-1-propanol (BAL) are known to induce starfish oocyte maturation [16]. However, microinjection of DTT into the immature oocytes of the starfish, Asterina pectinifera, failed to induce oocyte maturation, suggesting that the substance acts on the oocyte surface from the outside and indirectly induces oocyte maturation. Microinjection of the cytoplasm taken from the DTT-treated oocytes into immature oocytes was found to induce oocyte maturation, indicating that the cytoplasmic factor triggering the germinal vesicle breakdown and subsequent meiotic process is produced under the influence of DTT, as well as 1-methyladenine (1-MeAde). The pattern of changes in maturation-inducing capacity of the maturing cytoplasm of the DTT-treated oocytes was similar to that of the 1-MeAde-treated oocytes. Morphological changes in the oocyte surface and the vitelline coat of the DTT-treated oocytes were similar to those in the 1-MeAde-treated oocytes. The action of DTT in inducing maturation is considered to be the same as that of 1-MeAde. Sulfhydryl content of oocyte-cortex protein was found to increase after the treatment with 1-MeAde, suggesting that the action of 1-MeAde in inducing oocyte maturation is to be ascribed to its action of reducing disulfide bond.